This invention is in the field of diagnostic immunology. The invention provides means of identifying response of living patient cells to specific antigens as a means of identifying hypersensitivity and evaluating effects of regimens used to treat allergic responses. Another aspect of the invention is improved plates which are particularly useful for practice of the inventive process.
The evaluation of immune responses in patients presenting with clinical symptoms of allergic response presents many problems. While skin testing may be helpful, such testing has not provided an effective tool for evaluation of changes in immune response during treatment.
Since the 1960""s, numerous in vitro assays have been used to augment in vivo evaluation of immune response. Many of these bioassays are based on understanding of effect of antigenic substances on lymphocytes. Many of the tests evaluate lymphocyte proliferation in the presence of appropriately presented antigens require use of radioactive materials such as 3H-thymidine or [125I]iododeoxyuridine for evaluation of changes in cell activation. These assays require specialized equipment and Ye relatively expensive to perform. The tests of the prior art usually require 3-7 day""s time. Results are not as reliable as would be desired.
Ulf Landegren (J. Immunol. Meth. 67 379-388 (1984)) developed an assay to measure lymphocyte proliferation wherein he used a chromogenic indicator, p-nitrophenol-N-acetyl-xcex2-D-glucosaminide. The absorbance at 405 nm (A405), when read using a colorimetric plate reader, indicated the amount of color generated was directly proportional to the cell number.
Tim Mosmann (j. immunol. meth. 65: 55-63 (1983)) developed an assay using tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to measure mammalian cell survival and proliferation. The assay detects living cells. The response is dependent on the degree of activation of the cells. The method was used to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis. Using the tetrazolium salt as an indicator, it was found that the absorbance readings indicated a linear relationship between cell number and color formation.
Wilhelms, U.S. Pat. No. 4,592,997 teaches a method for evaluation of leukocyte activation by incubation of cells with allergens followed by measurement of protease liberation wherein a chromogenic substrate is used as the colorimetric indicator.
Hashimoto, et al. (J. Immunol. Meth. 90: 97-103 (1986)) taught use of an assay to detect B-cell proliferation using chromogenic indicators of intracellular enzymes such as APase. His teaching shows no evidence of activation prior to 3 days of exposure to the activating agent.
Chan, et al. (Anal. Biochem. 157: 375-380 (1986)) describes a direct colorimetric assay for measuring Ca2+-stimulated ed ATPase activity on adipocyte plasma membrane preparation. The enzyme was measured in cell extracts.